Computational Studies of Protein-Inhibitor Interactions

In this thesis work, I conducted large-scale molecular dynamics (MD) simulation studies of interactions of enzymes and signaling proteins with inhibitory ligands. Specifically, I have studied three classes of proteins: the first part of my thesis reports studies on the hydrogen-producing [FeFe]-hydrogenase enzyme, the second part reports on studies of regulatory proteins from the G-protein coupled receptor (GPCR) family, and the third part reports on studies of the phosphodiesterase (PDE) enzyme family. In the first part, I studied the problem of [FeFe]-hydrogenase sensitivity to the presence of inhibitory gases oxygen (O2) and carbon monoxide (CO) that cause irreversible damage to the active site of this enzyme. Therefore, a detailed knowledge of the diffusion pathways of these inhibitory gases is necessary to develop strategies for designing novel enzymes that are tolerant to these gases. Specifically, I studied the diffusion pathways of O2 and CO in the CpI [FeFe]-hydrogenase from Clostridium pasteurianum. I used several enhanced sampling and free-energy simulation methods to reconstruct a three-dimensional free-energy surface for diffusion of each gas which revealed free-energy minima forming an interconnected network of pathways. I discovered multiple pathways of minimal free-energy as diffusion portals for O2 and CO, and observed that the global minimum in the free-energy surface is located in the vicinity of the active site metal cluster, the H-cluster. Among potential residues that I propose as candidates for future mutagenesis studies to increase the tolerance of this enzyme to both inhibitory gases, 11 residues are shared between O2 and CO. I hypothesize that these shared candidate residues are potentially useful for designing new variants of this enzyme that are tolerant to both inhibitory gases. In the second part, I have studied the interplay of protein conformational dynamics and effects of small-molecule inhibitors in a class of signaling proteins, known as the Regulators of G protein signaling (RGS) proteins, that negatively modulate signaling in GPCRs. Recently discovered thiadiazolidinone(TDZD) compounds that target cysteine residues have shown different levels of specificities and potencies for several known RGS proteins, thereby suggesting intrinsic differences in dynamics of these proteins upon binding of these compounds. I characterized the effect of binding of several small-molecule inhibitors on perturbations and dynamical motions in RGS4. Specifically, I studied two conformational models of RGS4 in which a buried cysteine residue is solvent-exposed due to side-chain motions or due to flexibility in neighboring helices. I found that TDZD compounds with aromatic functional groups perturb the RGS4 structure more than compounds with aliphatic functional groups. Moreover, small-molecules with aromatic functional groups but lacking sulfur atoms only transiently reside within the protein and spontaneously dissociate to the solvent. I further probed the salt-bridges forming across isoforms of RGS proteins, resulting in a hypothesis that differences in salt-bridges between a pair of helices in RGS proteins are responsible for differences in flexibility and potency among isoforms. In the final part of my thesis, I evaluated differences in binding interactions of phosphodiesterase 4 (PDE4) inhibitors within the PDE4 catalytic domain. From residues within 5 Å of the ligand binding site, five residues revealed significant differences in non-bonded interaction energies that could account for the differential binding affinities of inhibitors. I found one site (Phe506 in human PDE4; Tyr253 in the C. elegans PDE4 catalytic domain) that alters the binding conformation of roflumilast and zardaverine (human PDE 4 inhibitors) into a less energetically favorable state. These results support the feasibility of designing the next-generation of anthelmintics/nematicides that could selectively bind to nematode PDEs. Overall, my thesis has resulted in enhancement of detailed mechanistic insights into several protein and inhibitory ligand interactions that are potentially useful in the developement of novel inhibitors targeting protein/protein and protein/ligand interactions

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

none2929siIn 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.openKlionsky, Daniel J; Abdel-Aziz, Amal Kamal; Abdelfatah, Sara; Abdellatif, Mahmoud; Abdoli, Asghar; Abel, Steffen; Abeliovich, Hagai; Abildgaard, Marie H; Abudu, Yakubu Princely; Acevedo-Arozena, Abraham; Adamopoulos, Iannis E; Adeli, Khosrow; Adolph, Timon E; Adornetto, Annagrazia; Aflaki, Elma; Agam, Galila; Agarwal, Anupam; Aggarwal, Bharat B; Agnello, Maria; Agostinis, Patrizia; Agrewala, Javed N; Agrotis, Alexander; Aguilar, Patricia V; Ahmad, S Tariq; Ahmed, Zubair M; Ahumada-Castro, Ulises; Aits, Sonja; Aizawa, Shu; Akkoc, Yunus; Akoumianaki, Tonia; Akpinar, Hafize Aysin; Al-Abd, Ahmed M; Al-Akra, Lina; Al-Gharaibeh, Abeer; Alaoui-Jamali, Moulay A; Alberti, Simon; Alcocer-Gómez, Elísabet; Alessandri, Cristiano; Ali, Muhammad; Alim Al-Bari, M Abdul; Aliwaini, Saeb; Alizadeh, Javad; Almacellas, Eugènia; Almasan, Alexandru; Alonso, Alicia; Alonso, Guillermo D; Altan-Bonnet, Nihal; Altieri, Dario C; Álvarez, Élida M C; Alves, Sara; Alves da Costa, Cristine; Alzaharna, Mazen M; Amadio, Marialaura; Amantini, Consuelo; Amaral, Cristina; Ambrosio, Susanna; Amer, Amal O; Ammanathan, Veena; An, Zhenyi; Andersen, Stig U; Andrabi, Shaida A; Andrade-Silva, Magaiver; Andres, Allen M; Angelini, Sabrina; Ann, David; Anozie, Uche C; Ansari, Mohammad Y; Antas, Pedro; Antebi, Adam; Antón, Zuriñe; Anwar, Tahira; Apetoh, Lionel; Apostolova, Nadezda; Araki, Toshiyuki; Araki, Yasuhiro; Arasaki, Kohei; Araújo, Wagner L; Araya, Jun; Arden, Catherine; Arévalo, Maria-Angeles; Arguelles, Sandro; Arias, Esperanza; Arikkath, Jyothi; Arimoto, Hirokazu; Ariosa, Aileen R; Armstrong-James, Darius; Arnauné-Pelloquin, Laetitia; Aroca, Angeles; Arroyo, Daniela S; Arsov, Ivica; Artero, Rubén; Asaro, Dalia Maria Lucia; Aschner, Michael; Ashrafizadeh, Milad; Ashur-Fabian, Osnat; Atanasov, Atanas G; Au, Alicia K; Auberger, Patrick; Auner, Holger W; Aurelian, Laure; Autelli, Riccardo; Avagliano, Laura; Ávalos, Yenniffer; Aveic, Sanja; Aveleira, Célia Alexandra; Avin-Wittenberg, Tamar; Aydin, Yucel; Ayton, Scott; Ayyadevara, Srinivas; Azzopardi, Maria; Baba, Misuzu; Backer, Jonathan M; Backues, Steven K; Bae, Dong-Hun; Bae, Ok-Nam; Bae, Soo Han; Baehrecke, Eric H; Baek, Ahruem; Baek, Seung-Hoon; Baek, Sung Hee; Bagetta, Giacinto; Bagniewska-Zadworna, Agnieszka; Bai, Hua; Bai, Jie; Bai, Xiyuan; Bai, Yidong; Bairagi, Nandadulal; Baksi, Shounak; Balbi, Teresa; Baldari, Cosima T; Balduini, Walter; Ballabio, Andrea; Ballester, Maria; Balazadeh, Salma; Balzan, Rena; Bandopadhyay, Rina; Banerjee, Sreeparna; Banerjee, Sulagna; Bánréti, Ágnes; Bao, Yan; Baptista, Mauricio S; Baracca, Alessandra; Barbati, Cristiana; Bargiela, Ariadna; Barilà, Daniela; Barlow, Peter G; Barmada, Sami J; Barreiro, Esther; Barreto, George E; Bartek, Jiri; Bartel, Bonnie; Bartolome, Alberto; Barve, Gaurav R; Basagoudanavar, Suresh H; Bassham, Diane C; Bast, Robert C; Basu, Alakananda; Batoko, Henri; Batten, Isabella; Baulieu, Etienne E; Baumgarner, Bradley L; Bayry, Jagadeesh; Beale, Rupert; Beau, Isabelle; Beaumatin, Florian; Bechara, Luiz R G; Beck, George R; Beers, Michael F; Begun, Jakob; Behrends, Christian; Behrens, Georg M N; Bei, Roberto; Bejarano, Eloy; Bel, Shai; Behl, Christian; Belaid, Amine; Belgareh-Touzé, Naïma; Bellarosa, Cristina; Belleudi, Francesca; Belló Pérez, Melissa; Bello-Morales, Raquel; Beltran, Jackeline Soares de Oliveira; Beltran, Sebastián; Benbrook, Doris Mangiaracina; Bendorius, Mykolas; Benitez, Bruno A; Benito-Cuesta, Irene; Bensalem, Julien; Berchtold, Martin W; Berezowska, Sabina; Bergamaschi, Daniele; Bergami, Matteo; Bergmann, Andreas; Berliocchi, Laura; Berlioz-Torrent, Clarisse; Bernard, Amélie; Berthoux, Lionel; Besirli, Cagri G; Besteiro, Sebastien; Betin, Virginie M; Beyaert, Rudi; Bezbradica, Jelena S; Bhaskar, Kiran; Bhatia-Kissova, Ingrid; Bhattacharya, Resham; Bhattacharya, Sujoy; Bhattacharyya, Shalmoli; Bhuiyan, Md Shenuarin; Bhutia, Sujit Kumar; Bi, Lanrong; Bi, Xiaolin; Biden, Trevor J; Bijian, Krikor; Billes, Viktor A; Binart, Nadine; Bincoletto, Claudia; Birgisdottir, Asa B; Bjorkoy, Geir; Blanco, Gonzalo; Blas-Garcia, Ana; Blasiak, Janusz; Blomgran, Robert; Blomgren, Klas; Blum, Janice S; Boada-Romero, Emilio; Boban, Mirta; Boesze-Battaglia, Kathleen; Boeuf, Philippe; Boland, Barry; Bomont, Pascale; Bonaldo, Paolo; Bonam, Srinivasa Reddy; Bonfili, Laura; Bonifacino, Juan S; Boone, Brian A; Bootman, Martin D; Bordi, Matteo; Borner, Christoph; Bornhauser, Beat C; Borthakur, Gautam; Bosch, Jürgen; Bose, Santanu; Botana, Luis M; Botas, Juan; Boulanger, Chantal M; Boulton, Michael E; Bourdenx, Mathieu; Bourgeois, Benjamin; Bourke, Nollaig M; Bousquet, Guilhem; Boya, Patricia; Bozhkov, Peter V; Bozi, Luiz H M; Bozkurt, Tolga O; Brackney, Doug E; Brandts, Christian H; Braun, Ralf J; Braus, Gerhard H; Bravo-Sagua, Roberto; Bravo-San Pedro, José M; Brest, Patrick; Bringer, Marie-Agnès; Briones-Herrera, Alfredo; Broaddus, V Courtney; Brodersen, Peter; Brodsky, Jeffrey L; Brody, Steven L; Bronson, Paola G; Bronstein, Jeff M; Brown, Carolyn N; Brown, Rhoderick E; Brum, Patricia C; Brumell, John H; Brunetti-Pierri, Nicola; Bruno, Daniele; Bryson-Richardson, Robert J; Bucci, Cecilia; Buchrieser, Carmen; Bueno, Marta; Buitrago-Molina, Laura Elisa; Buraschi, Simone; Buch, Shilpa; Buchan, J Ross; Buckingham, Erin M; Budak, Hikmet; Budini, Mauricio; Bultynck, Geert; Burada, Florin; Burgoyne, Joseph R; Burón, M Isabel; Bustos, Victor; Büttner, Sabrina; Butturini, Elena; Byrd, Aaron; Cabas, Isabel; Cabrera-Benitez, Sandra; Cadwell, Ken; Cai, Jingjing; Cai, Lu; Cai, Qian; Cairó, Montserrat; Calbet, Jose A; Caldwell, Guy A; Caldwell, Kim A; Call, Jarrod A; Calvani, Riccardo; Calvo, Ana C; Calvo-Rubio Barrera, Miguel; Camara, Niels Os; Camonis, Jacques H; Camougrand, Nadine; Campanella, Michelangelo; Campbell, Edward M; Campbell-Valois, François-Xavier; Campello, Silvia; Campesi, Ilaria; Campos, Juliane C; Camuzard, Olivier; Cancino, Jorge; Candido de Almeida, Danilo; Canesi, Laura; Caniggia, Isabella; Canonico, Barbara; Cantí, Carles; Cao, Bin; Caraglia, Michele; Caramés, Beatriz; Carchman, Evie H; Cardenal-Muñoz, Elena; Cardenas, Cesar; Cardenas, Luis; Cardoso, Sandra M; Carew, Jennifer S; Carle, Georges F; Carleton, Gillian; Carloni, Silvia; Carmona-Gutierrez, Didac; Carneiro, Leticia A; Carnevali, Oliana; Carosi, Julian M; Carra, Serena; Carrier, Alice; Carrier, Lucie; Carroll, Bernadette; Carter, A Brent; Carvalho, Andreia Neves; Casanova, Magali; Casas, Caty; Casas, Josefina; Cassioli, Chiara; Castillo, Eliseo F; Castillo, Karen; Castillo-Lluva, Sonia; Castoldi, Francesca; Castori, Marco; Castro, Ariel F; Castro-Caldas, Margarida; Castro-Hernandez, Javier; Castro-Obregon, Susana; Catz, Sergio D; Cavadas, Claudia; Cavaliere, Federica; Cavallini, Gabriella; Cavinato, Maria; Cayuela, Maria L; Cebollada Rica, Paula; Cecarini, Valentina; Cecconi, Francesco; Cechowska-Pasko, Marzanna; Cenci, Simone; Ceperuelo-Mallafré, Victòria; Cerqueira, João J; Cerutti, Janete M; Cervia, Davide; Cetintas, Vildan Bozok; Cetrullo, Silvia; Chae, Han-Jung; Chagin, Andrei S; Chai, Chee-Yin; Chakrabarti, Gopal; Chakrabarti, Oishee; Chakraborty, Tapas; Chakraborty, Trinad; Chami, Mounia; Chamilos, Georgios; Chan, David W; Chan, Edmond Y W; Chan, Edward D; Chan, H Y Edwin; Chan, Helen H; Chan, Hung; Chan, Matthew T V; Chan, Yau Sang; Chandra, Partha K; Chang, Chih-Peng; Chang, Chunmei; Chang, Hao-Chun; Chang, Kai; Chao, Jie; Chapman, Tracey; Charlet-Berguerand, Nicolas; Chatterjee, Samrat; Chaube, Shail K; Chaudhary, Anu; Chauhan, Santosh; Chaum, Edward; Checler, Frédéric; Cheetham, Michael E; Chen, Chang-Shi; Chen, Guang-Chao; Chen, Jian-Fu; Chen, Liam L; Chen, Leilei; Chen, Lin; Chen, Mingliang; Chen, Mu-Kuan; Chen, Ning; Chen, Quan; Chen, Ruey-Hwa; Chen, Shi; Chen, Wei; Chen, Weiqiang; Chen, Xin-Ming; Chen, Xiong-Wen; Chen, Xu; Chen, Yan; Chen, Ye-Guang; Chen, Yingyu; Chen, Yongqiang; Chen, Yu-Jen; Chen, Yue-Qin; Chen, Zhefan Stephen; Chen, Zhi; Chen, Zhi-Hua; Chen, Zhijian J; Chen, Zhixiang; Cheng, Hanhua; Cheng, Jun; Cheng, Shi-Yuan; Cheng, Wei; Cheng, Xiaodong; Cheng, Xiu-Tang; Cheng, Yiyun; Cheng, Zhiyong; Chen, Zhong; Cheong, Heesun; Cheong, Jit Kong; Chernyak, Boris V; Cherry, Sara; Cheung, Chi Fai Randy; Cheung, Chun Hei Antonio; Cheung, King-Ho; Chevet, Eric; Chi, Richard J; Chiang, Alan Kwok Shing; Chiaradonna, Ferdinando; Chiarelli, Roberto; Chiariello, Mario; Chica, Nathalia; Chiocca, Susanna; Chiong, Mario; Chiou, Shih-Hwa; Chiramel, Abhilash I; Chiurchiù, Valerio; Cho, Dong-Hyung; Choe, Seong-Kyu; Choi, Augustine M K; Choi, Mary E; Choudhury, Kamalika Roy; Chow, Norman S; Chu, Charleen T; Chua, Jason P; Chua, John Jia En; Chung, Hyewon; Chung, Kin Pan; Chung, Seockhoon; Chung, So-Hyang; Chung, Yuen-Li; Cianfanelli, Valentina; Ciechomska, Iwona A; Cifuentes, Mariana; Cinque, Laura; Cirak, Sebahattin; Cirone, Mara; Clague, Michael J; Clarke, Robert; Clementi, Emilio; Coccia, Eliana M; Codogno, Patrice; Cohen, Ehud; Cohen, Mickael M; Colasanti, Tania; Colasuonno, Fiorella; Colbert, Robert A; Colell, Anna; Čolić, Miodrag; Coll, Nuria S; Collins, Mark O; Colombo, María I; Colón-Ramos, Daniel A; Combaret, Lydie; Comincini, Sergio; Cominetti, Márcia R; Consiglio, Antonella; Conte, Andrea; Conti, Fabrizio; Contu, Viorica Raluca; Cookson, Mark R; Coombs, Kevin M; Coppens, Isabelle; Corasaniti, Maria Tiziana; Corkery, Dale P; Cordes, Nils; Cortese, Katia; Costa, Maria do Carmo; Costantino, Sarah; Costelli, Paola; Coto-Montes, Ana; Crack, Peter J; Crespo, Jose L; Criollo, Alfredo; Crippa, Valeria; Cristofani, Riccardo; Csizmadia, Tamas; Cuadrado, Antonio; Cui, Bing; Cui, Jun; Cui, Yixian; Cui, Yong; Culetto, Emmanuel; Cumino, Andrea C; Cybulsky, Andrey V; Czaja, Mark J; Czuczwar, Stanislaw J; D'Adamo, Stefania; D'Amelio, Marcello; D'Arcangelo, Daniela; D'Lugos, Andrew C; D'Orazi, Gabriella; da Silva, James A; Dafsari, Hormos Salimi; Dagda, Ruben K; Dagdas, Yasin; Daglia, Maria; Dai, Xiaoxia; Dai, Yun; Dai, Yuyuan; Dal Col, Jessica; Dalhaimer, Paul; Dalla Valle, Luisa; Dallenga, Tobias; Dalmasso, Guillaume; Damme, Markus; Dando, Ilaria; Dantuma, Nico P; Darling, April L; Das, Hiranmoy; Dasarathy, Srinivasan; Dasari, Santosh K; Dash, Srikanta; Daumke, Oliver; Dauphinee, Adrian N; Davies, Jeffrey S; Dávila, Valeria A; Davis, Roger J; Davis, Tanja; Dayalan Naidu, Sharadha; De Amicis, Francesca; De Bosscher, Karolien; De Felice, Francesca; De Franceschi, Lucia; De Leonibus, Chiara; de Mattos Barbosa, Mayara G; De Meyer, Guido R Y; De Milito, Angelo; De Nunzio, Cosimo; De Palma, Clara; De Santi, Mauro; De Virgilio, Claudio; De Zio, Daniela; Debnath, Jayanta; DeBosch, Brian J; Decuypere, Jean-Paul; Deehan, Mark A; Deflorian, Gianluca; DeGregori, James; Dehay, Benjamin; Del Rio, Gabriel; Delaney, Joe R; Delbridge, Lea M D; Delorme-Axford, Elizabeth; Delpino, M Victoria; Demarchi, Francesca; Dembitz, Vilma; Demers, Nicholas D; Deng, Hongbin; Deng, Zhiqiang; Dengjel, Joern; Dent, Paul; Denton, Donna; DePamphilis, Melvin L; Der, Channing J; Deretic, Vojo; Descoteaux, Albert; Devis, Laura; Devkota, Sushil; Devuyst, Olivier; Dewson, Grant; Dharmasivam, Mahendiran; Dhiman, Rohan; di Bernardo, Diego; Di Cristina, Manlio; Di Domenico, Fabio; Di Fazio, Pietro; Di Fonzo, Alessio; Di Guardo, Giovanni; Di Guglielmo, Gianni M; Di Leo, Luca; Di Malta, Chiara; Di Nardo, Alessia; Di Rienzo, Martina; Di Sano, Federica; Diallinas, George; Diao, Jiajie; Diaz-Araya, Guillermo; Díaz-Laviada, Inés; Dickinson, Jared M; Diederich, Marc; Dieudé, Mélanie; Dikic, Ivan; Ding, Shiping; Ding, Wen-Xing; Dini, Luciana; Dinić, Jelena; Dinic, Miroslav; Dinkova-Kostova, Albena T; Dionne, Marc S; Distler, Jörg H W; Diwan, Abhinav; Dixon, Ian M C; Djavaheri-Mergny, Mojgan; Dobrinski, Ina; Dobrovinskaya, Oxana; Dobrowolski, Radek; Dobson, Renwick C J; Đokić, Jelena; Dokmeci Emre, Serap; Donadelli, Massimo; Dong, Bo; Dong, Xiaonan; Dong, Zhiwu; Dorn Ii, Gerald W; Dotsch, Volker; Dou, Huan; Dou, Juan; Dowaidar, Moataz; Dridi, Sami; Drucker, Liat; Du, Ailian; Du, Caigan; Du, Guangwei; Du, Hai-Ning; Du, Li-Lin; du Toit, André; Duan, Shao-Bin; Duan, Xiaoqiong; Duarte, Sónia P; Dubrovska, Anna; Dunlop, Elaine A; Dupont, Nicolas; Durán, Raúl V; Dwarakanath, Bilikere S; Dyshlovoy, Sergey A; Ebrahimi-Fakhari, Darius; Eckhart, Leopold; Edelstein, Charles L; Efferth, Thomas; Eftekharpour, Eftekhar; Eichinger, Ludwig; Eid, Nabil; Eisenberg, Tobias; Eissa, N Tony; Eissa, Sanaa; Ejarque, Miriam; El Andaloussi, Abdeljabar; El-Hage, Nazira; El-Naggar, Shahenda; Eleuteri, Anna Maria; El-Shafey, Eman S; Elgendy, Mohamed; Eliopoulos, Aristides G; Elizalde, María M; Elks, Philip M; Elsasser, Hans-Peter; Elsherbiny, Eslam S; Emerling, Brooke M; Emre, N C Tolga; Eng, Christina H; Engedal, Nikolai; Engelbrecht, Anna-Mart; Engelsen, Agnete S T; Enserink, Jorrit M; Escalante, Ricardo; Esclatine, Audrey; Escobar-Henriques, Mafalda; Eskelinen, Eeva-Liisa; Espert, Lucile; Eusebio, Makandjou-Ola; Fabrias, Gemma; Fabrizi, Cinzia; Facchiano, Antonio; Facchiano, Francesco; Fadeel, Bengt; Fader, Claudio; Faesen, Alex C; Fairlie, W Douglas; Falcó, Alberto; Falkenburger, Bjorn H; Fan, Daping; Fan, Jie; Fan, Yanbo; Fang, Evandro F; Fang, Yanshan; Fang, Yognqi; Fanto, Manolis; Farfel-Becker, Tamar; Faure, Mathias; Fazeli, Gholamreza; Fedele, Anthony O; Feldman, Arthur M; Feng, Du; Feng, Jiachun; Feng, Lifeng; Feng, Yibin; Feng, Yuchen; Feng, Wei; Fenz Araujo, Thais; Ferguson, Thomas A; Fernández, Álvaro F; Fernandez-Checa, Jose C; Fernández-Veledo, Sonia; Fernie, Alisdair R; Ferrante, Anthony W; Ferraresi, Alessandra; Ferrari, Merari F; Ferreira, Julio C B; Ferro-Novick, Susan; Figueras, Antonio; Filadi, Riccardo; Filigheddu, Nicoletta; Filippi-Chiela, Eduardo; Filomeni, Giuseppe; Fimia, Gian Maria; Fineschi, Vittorio; Finetti, Francesca; Finkbeiner, Steven; Fisher, Edward A; Fisher, Paul B; Flamigni, Flavio; Fliesler, Steven J; Flo, Trude H; Florance, Ida; Florey, Oliver; Florio, Tullio; Fodor, Erika; Follo, Carlo; Fon, Edward A; Forlino, Antonella; Fornai, Francesco; Fortini, Paola; Fracassi, Anna; Fraldi, Alessandro; Franco, Brunella; Franco, Rodrigo; Franconi, Flavia; Frankel, Lisa B; Friedman, Scott L; Fröhlich, Leopold F; Frühbeck, Gema; Fuentes, Jose M; Fujiki, Yukio; Fujita, Naonobu; Fujiwara, Yuuki; Fukuda, Mitsunori; Fulda, Simone; Furic, Luc; Furuya, Norihiko; Fusco, Carmela; Gack, Michaela U; Gaffke, Lidia; Galadari, Sehamuddin; Galasso, Alessia; Galindo, Maria F; Gallolu Kankanamalage, Sachith; Galluzzi, Lorenzo; Galy, Vincent; Gammoh, Noor; Gan, Boyi; Ganley, Ian G; Gao, Feng; Gao, Hui; Gao, Minghui; Gao, Ping; Gao, Shou-Jiang; Gao, Wentao; Gao, Xiaobo; Garcera, Ana; Garcia, Maria Noé; Garcia, Verónica E; García-Del Portillo, Francisco; Garcia-Escudero, Vega; Garcia-Garcia, Aracely; Garcia-Macia, Marina; García-Moreno, Diana; Garcia-Ruiz, Carmen; García-Sanz, Patricia; Garg, Abhishek D; Gargini, Ricardo; Garofalo, Tina; Garry, Robert F; Gassen, Nils C; Gatica, Damian; Ge, Liang; Ge, Wanzhong; Geiss-Friedlander, Ruth; Gelfi, Cecilia; Genschik, Pascal; Gentle, Ian E; Gerbino, Valeria; Gerhardt, Christoph; Germain, Kyla; Germain, Marc; Gewirtz, David A; Ghasemipour Afshar, Elham; Ghavami, Saeid; Ghigo, Alessandra; Ghosh, Manosij; Giamas, Georgios; Giampietri, Claudia; Giatromanolaki, Alexandra; Gibson, Gary E; Gibson, Spencer B; Ginet, Vanessa; Giniger, Edward; Giorgi, Carlotta; Girao, Henrique; Girardin, Stephen E; Giridharan, Mridhula; Giuliano, Sandy; Giulivi, Cecilia; Giuriato, Sylvie; Giustiniani, Julien; Gluschko, Alexander; Goder, Veit; Goginashvili, Alexander; Golab, Jakub; Goldstone, David C; Golebiewska, Anna; Gomes, Luciana R; Gomez, Rodrigo; Gómez-Sánchez, Rubén; Gomez-Puerto, Maria Catalina; Gomez-Sintes, Raquel; Gong, Qingqiu; Goni, Felix M; González-Gallego, Javier; Gonzalez-Hernandez, Tomas; Gonzalez-Polo, Rosa A; Gonzalez-Reyes, Jose A; González-Rodríguez, Patricia; Goping, Ing Swie; Gorbatyuk, Marina S; Gorbunov, Nikolai V; Görgülü, Kıvanç; Gorojod, Roxana M; Gorski, Sharon M; Goruppi, Sandro; Gotor, Cecilia; Gottlieb, Roberta A; Gozes, Illana; Gozuacik, Devrim; Graef, Martin; Gräler, Markus H; Granatiero, Veronica; Grasso, Daniel; Gray, Joshua P; Green, Douglas R; Greenhough, Alexander; Gregory, Stephen L; Griffin, Edward F; Grinstaff, Mark W; Gros, Frederic; Grose, Charles; Gross, Angelina S; Gruber, Florian; Grumati, Paolo; Grune, Tilman; Gu, Xueyan; Guan, Jun-Lin; Guardia, Carlos M; Guda, Kishore; Guerra, Flora; Guerri, Consuelo; Guha, Prasun; Guillén, Carlos; Gujar, Shashi; Gukovskaya, Anna; Gukovsky, Ilya; Gunst, Jan; Günther, Andreas; Guntur, Anyonya R; Guo, Chuanyong; Guo, Chun; Guo, Hongqing; Guo, Lian-Wang; Guo, Ming; Gupta, Pawan; Gupta, Shashi Kumar; Gupta, Swapnil; Gupta, Veer Bala; Gupta, Vivek; Gustafsson, Asa B; Gutterman, David D; H B, Ranjitha; Haapasalo, Annakaisa; Haber, James E; Hać, Aleksandra; Hadano, Shinji; Hafrén, Anders J; Haidar, Mansour; Hall, Belinda S; Halldén, Gunnel; Hamacher-Brady, Anne; Hamann, Andrea; Hamasaki, Maho; Han, Weidong; Hansen, Malene; Hanson, Phyllis I; Hao, Zijian; 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Guidelines for the use and interpretation of assays for monitoring autophagy (2nd edition)

In 2008 we published the first set of guidelines for standardiz- ing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new tech- nologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in differ- ent organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes..

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field